Aortic histology and immunohistochemistry quantification

Mice dissections were done as previously described (42). Briefly, following dissection, hearts were either transferred to 70% ethanol and stored at 4°C for future creation of formalin-fixed, paraffin-embedded sections or transferred to 30% sterile sucrose for 72 h for generating cryosectioning slides. Formalin-fixed, paraffin-embedded sections were stained with Masson’s trichrome for quantification of collagen content, necrotic core areas, and subendothelium cell numbers, and with anti-MOMA2 for MΦs. Frozen sections were stained with Oil Red O (ORO) for quantification of lesion area. A color deconvolution algorithm, developed by UNC Translational Pathology Laboratory, was used for digital quantification of collagen, reported as OD × percent of total positive area stained with collagen (44–46). Necrotic core size and subendothelial cell nuclei were quantified on Masson’s trichrome–stained slides, which were normalized to the total quantified area. Necrotic core size was measured using Aperio ePathology software (Aperio, Buffalo Grove, IL), and subendothelial cell numbers were assessed manually by counting nuclei. Quantification of MΦs was calculated as OD × percent of total positive area stained with anti-MOMA2. Quantification of atherosclerotic lesions was done as described with modification (47, 48).